We have recently discovered that the cationic lipids DOTAP and DOEPC, originally developed as transfection reagents, can deliver a peptide antigen E7 to the dendritic cells (DC) and illicit a potent CTL activity against TC-1 tumor cells expressing the antigen. Signal transduction studies in the primary bone marrow-derived DC (BMDC) indicate that reactive oxygen species (ROS) is stimulated by DOTAP which leads to the phosphorylation and activation of two MAP kinases, ERK and p38,. pERK activation leads to the production and the secretion of potent chemokines, CCL2 and CCL4, by BMDC. P38 activation leads to the production of IL-12. Inhibition of pERK activation by specific inhibitors of Mek1/2, the upstream kinase which phosphorylates ERK, inhibited CCL2 production and, more importantly, the antitumor activity of DOTAP/E7. We hypothesize that ERK and p38 activation play a key role in the adjuvant activity of DOTAP, perhaps also DOEPC. The project will study in aim 1 the signaling of ROS and its downstream events leading to the production of CCL2 and IL-12. In aim 2, we will perform an extensive structure-activity relationship study of the cationic lipid to identify more active species that are suitable for vaccine formulation. Various DOTAP analogs will be synthesized and tested for ROS activation and vaccine activity. Finally in aim 3, we will attempt to improve the anti-tumor vaccine activity using the TC-1 tumor as a model for human cervical cancer. A number of formulation improvement strategies will be tested in the model. Furthermore, we will test the activity of siRNA against VEGF and other key oncogenes delivered by a targeted nanoparticle formulation to enhance the anti- tumor activity of the DOTAP/E7 vaccine. The goal of the study is to develop chemically well defined and potent cationic lipids for therapeutic vaccines against cancer. PUBLIC HEALTH RELEVANCE: The goal of the project is to develop an effective therapeutic vaccine for cervical cancer. The vaccine contains only two molecules, the antigen and a lipid, and is a very simple but highly active vaccine in a mouse model. Project will study the activity and the mechanism of the lipid adjuvant, in order to find more active lipid and more efficacious vaccine.